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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
  • C12G1/00—Preparation of wine or sparkling wine
  • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
  • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/36—Adaptation or attenuation of cells

Definitions

• TITLE Lactobacillus plantarum cells with improved resistance to high concentrations of ethanol .
• the present invention relates to cycloserine resistant mutants of lactic acid bacteria characterized by having improved resistance towards ethanol .
• the cycloserine resistant mutants of lactic acid bacteria can e.g . be used for malolactic fermentations of wine (i.e. including sparkling wine such as Cava/champagne) having high alcohol levels.
• Lactic acid bacteria such as Lactobacillus plantarum and Oenococcus oeni are used in the wine industry for malolactic fermentation . Their functionality is based on the ability to convert malic acid into the gentler lactic acid which is used for many types of wine.
• the malolactic fermentation is produced by means of the spontaneous growth of an indigenous flora of lactic acid bacteria.
• the process of MLF begins of its own accord, when the malolactic flora is sufficiently developed, that is to say in a random manner between the end of alcoholic fermentation and several weeks, even several months, after the alcoholic fermentation.
• the malolactic bacteria reach a con- centration of about 10 s CFU/ml in the medium, they enter an active metabolic phase and start the fermentation of the malic acid .
• Oenococcus oeni is the species most frequently responsible for the MLF.
• US7625745B2 (PCT filed 2004 and published in 2009) describes the selection of alco- hol-resistant Lactobacillus plantarum lactic acid bacterial strains and it is said that the authors believed that it was unexpected that it was possible to select such alcohol-resistant Lactobacillus plantarum lactic acid bacterial strains - e.g. due to that this resistance to alcohol was hitherto unknown for such the Lactobacillus plantarum strains (see e.g . C4, I . 20-30 and figures of US7625745B2).
• the isolates of natural lactic acid bacteria were subjected to a selection pressure of resistance to alcohol levels above 10% and two particular L. plantarum strains were found to be sufficiently resistant to alcohol levels above 10% (see e.g . Example 7 of US7625745B2) .
• D-cycloserine (D-4-amino-isoxazolidone) is an antibiotic which inhibits alanine race- mase, D-alanyl-D-alanine ligase, D-alanylalanine synthase and D-alanine permease causing cell lysis.
• D-alanine racemase is essential for the production of D-alanine, an integral part of the peptidoglycan layer of the cell wall .
• Strains of Lactobacillus plantarum in which the alanine racemase gene ⁇ air) of Lacto- coccus lactis has been inserted on a plasmid have resistance to D-cycloserine (Bron et al., 2002 Appl Environ Microbiol . 68 : 5663-5670).
• the problem to be solved by the present invention is to provide a Lactobacillus plantarum composition with improved resistance towards for wine relatively high concentrations of ethanol.
• the Lactobacillus plantarum composition is particularly useful for production of wine.
• the solution is based on that the present inventors have developed a novel selection method for the identification of new improved Lactobacillus plantarum compositions.
• a novel important step of the herein described new selection method relates to that the present inventors have identified a herein surprisingly relevant link between increased resistance to D-cycloserine and improved resistance to high concentrations of ethanol.
• novel selection method may overall be seen as comprising following two steps:
• step (ii) from the pool of D-cycloserine resistant cells identified in step (i) is then screened/selected for a Lactobacillus plantarum strain/cell that has improved resistance to a for wine relatively high concentrations of ethanol .
• the present inventors identified that from a pool of D-cycloserine resistant selected Lactobacillus plantarum strains/cells (i .e. from step (i) above) was it relatively rapid to then screen/select for a Lactobacillus plantarum strain/cell that has improved resistance to relatively high concentrations of ethanol - essentially, the reason for this is that the presents inventors surprisingly identified that a relatively high percentage of the first selected D- cycloserine resistant cells were also resistant to relatively high concentrations of ethanol .
• the first screening/selection for increased D-cycloserine resistance may, as discussed herein, be seen as a kind of pre-step to rapidly and efficient be able to screen/select/enrich for a Lactobacillus plantarum strain/cell that has improved resistance ethanol.
• a significant advantage of the herein described screening/selection method is that one relatively rapidly and efficiently is able to screen/select for a Lactobacillus plantarum strain/cell that has improved resistance ethanol.
• Lactobacillus plantarum strain with commercial relevant good properties in relation to e.g . malolactic fermentation of wine grape juice - one can then use this strain as a starting cell for mutagenesis and then relatively rapidly select for and thereby get/identify a novel Lactobacillus plantarum strain that has improved resistance to ethanol and still maintains its earlier good properties with respect to e.g . malolactic fermentation of wine grape juice.
• Lactobacillus plantarum cells are first selected for increased resistance to D-cycloserine - one may term it as resistance to D- cycloserine that is significantly higher than normally present in natural/wildtype Lactobacillus plantarum strains.
• a first aspect of the invention relates to a Lactobacillus plantarum composition, which comprises from 10 4 to 10 14 CFU/g Lactobacillus plantarum cells, wherein the Lactobacillus plantarum composition is characterized by that:
• the Lactobacillus plantarum cells have an increased resistance to D-cycloserine - defined by that the cells are Lactobacillus plantarum cells, wherein the amount of D- cycloserine that reduces the OD 6 oo measured growth, after 24 hours growth at 18°C, with 50% in the known Grape Juice GJ-5 medium
• GJ-5 medium has the following composition : Grape juice concentrate 70.0 g, Yeast paste 30.0 g. Tween 80 0.5 g, MnS04H20 0.1 g and Tap water 900.0 g) as compared to the growth in the GJ-5 medium without D-cycloserine (i .e. with 0 g/ml D-cycloserine) is higher than 70 g/ml D-cycloserine; and
• Lactobacillus plantarum cells have an improved resistance towards ethanol - defined by that the cells are Lactobacillus plantarum cells, wherein the cells can grow to an OD 50 o of at least 0.8 after 3 days incubation at 25°C in the GJ-5 medium with 11% ethanol .
• Lactobacillus planta- rum composition of the first aspect herein is a composition, wherein
• the cells in point (i) of first aspect) are positively resistant to D-cycloserine in the D-cycloserine resistance assay of example 1 ;
• Both the D-cycloserine resistance assay [of point (i)] and ethanol resistance assay [of point (ii)] are based on known, commercially available standard elements (such as e.g . standard media etc) . Accordingly, based on the detailed assay description herein (see e.g . example 1 herein for D-cycloserine resistance assay and example 2 herein for ethanol resistance assay) the skilled person is routinely able to repeat these assays to objectively determine whether a specific Lactobacillus plantarum cell of interest complies with the D-cycloserine resistance [of point (i)] and ethanol resistance [of point (ii)] levels of the first aspect of the invention.
• novel Lactobacillus plantarum composition as described herein may preferably be used for wine production.
• the dose and administration may be done according to the art.
• a second aspect of the invention relates to a method for producing a wine comprising administering the Lactobacillus plantarum composition of first aspect and herein described related embodiments to a grape juice or wine and performing further adequate steps to make the wine.
• a third aspect of the invention relates to a method for screening and isolating a novel Lactobacillus plantarum cell comprising the following steps:
• step (a) selecting and isolating from a pool of individual Lactobacillus plantarum cells, a new selected pool of Lactobacillus plantarum cells that have increased resistance to D-cycloserine under the conditions of point (i) of first aspect;
• step (b) selecting and isolating - from the selected pool of Lactobacillus plantarum D-cycloserine resistant cells of step (a) - a new isolated Lactobacillus plantarum cell that has improved resistance towards ethanol under the conditions of point (ii) of first aspect.
• Lactoba- cillus plantarum is a well know term to the skilled person and the skilled person knows if a particular lactic acid bacterium cell of interest is a Lactoba- cillus plantarum cell or not.
• DRAWINGS Figure 1 In this figure the resistance to D-cycloserine of different Lactobacillus plantarum strains is shown : For further details - see working Examples herein.
• Lactobacillus plantarum composition Lactobacillus plantarum composition
• Lactobacillus plantarum composition shall be understood according to the art. It is herein understood as a Lactobacillus plantarum composition comprising a number of Lactobacillus plantarum cells with a characteristic of interest.
• the Lactobacillus plantarum composition may comprise different types or strains of Lactobacillus plantarum cells (e.g. the two different Lactobacillus plantarum CHCC14254 and CHCC14255 strains discussed herein). In essence the composition shall simply comprise the amount of Lactobacillus plantarum cells given in the first aspect herein, wherein the Lactobacillus plantarum cells comply with the criteria given in the first aspect.
• commercially relevant Lactobacillus plantarum cell compositions are generally made by fermentation.
• the obtained Lactobacillus plantarum cells are generally concentrated, dried, mixed with a carrier and packed into a suitable container.
• the relevant e.g . 10 4 to 10 14 CFU/g Lactobacillus plantarum cells of the composition may be present in a commercially relevant form known to the skilled person.
• CFU/g Lactobacillus plantarum cells of the composition are present as dried (e.g . spray dried) cells or as frozen cells.
• the Lactobacillus plantarum composition comprises from 10 s to 10 14 CFU/g Lactobacillus plantarum cells, more preferably from 10 8 to 10 14 CFU/g Lactobacillus plantarum cells.
• CFU/g relates to the gram weight of the composition as such, including suitable relevant additives present in the composition . It does not include the weight of a suitable container used to package the Lactobacillus plantarum composition .
• An embodiment relates to that the Lactobacillus plantarum composition is packaged into a suitable container.
• a commercially relevant bacterial composition generally also comprises other relevant suitable additives. Beside the herein relevant Lactobacillus plantarum cells the composition may also comprise other relevant microorganisms of interest such as e.g . other lactic acid bacteria of interest or yeast cells of interest (such as e.g . wine yeast cells of interest) . Assay to select for an increased resistance to D-cycloserine
• D-cycloserine resistance assay of point (i) of first aspect is based on known commercially available standard elements (such as e.g . standard media, etc).
• the level of resistance as required in the assay of example 1 is a resistance to D-cycloserine that is significantly higher than normally present in natural/wildtype Lactobacillus plantarum strains.
• example 1 is herein a preferred assay to determine if a Lactobacillus plantarum cell of interest complies with the criteria of point (i) of first aspect. Increased resistance to D-cycloserine - point (i) of first aspect
• the increased resistance to D-cycloserine is higher than the one given in point (i) of the first aspect herein. Accordingly, it may be preferred that the Lactobacillus plantarum cells have an increased resistance to D-cycloserine - defined by that the cells are Lactobacillus plantarum cells, wherein the amount of D-cycloserine that reduces the OD 50 o measured growth, after 24 hours growth at 18°C, with 50% in the known Grape Juice GJ-5 medium as compared to the growth in the GJ-5 medium without D-cycloserine (i .e. with 0 pg/ml D-cycloserine) is higher than 80 g/ml D-cycloserine or is higher than 90 g/ml D-cycloserine.
• the ethanol resistance assay of point (ii) of first aspect is based on known commercially available standard elements (such as e.g. standard media, etc) . Accordingly, based on the detailed assay description herein (see e.g . example 2 herein) the skilled person is routinely able to repeat this assay to objectively determine whether a specific cell of interest complies with the ethanol resistance criteria as described in point (ii) .
• example 2 herein a preferred assay to determine if a Lactobacillus plantarum cell of interest complies with the criteria of point (ii) of first aspect. Improved resistance towards ethanol - point (ii) of first aspect
• the improved resistance towards ethanol is higher than the one given in point (ii) of the first aspect herein.
• the Lactobacillus plantarum cells have an im- proved resistance towards ethanol - defined by that the cells are Lactobacillus plantarum cells, wherein the cells can grow to an OD 6 oo of at least 0.8 after 3 days incubation at 25°C in the GJ-5 medium with 11.5% ethanol or with 12% ethanol or with 13% ethanol .
• a second aspect of the invention relates to a method for producing a wine comprising administering the Lactobacillus plantarum composition of first aspect and herein described related embodiments to a grape juice or wine and per- forming further adequate steps to make the wine.
• the wine may be any wine of interest such as red wine, white wine or sparkling wine such as Cava/champagne.
• red wine white wine
• sparkling wine such as Cava/champagne.
• Lactobacillus plantarum cells 10 5 CFU per ml grape juice or wine.
• a method for cocoa bean fermentation As known in the art - Lactobacillus plantarum cells have been used for cocoa bean fermentation. In line of this - a herein relevant use of the Lactobacillus plantarum cells as described herein is use for cocoa bean fermentation - i.e. a method for cocoa bean fermentation, wherein a Lactobacillus plantarum composition as described herein is inoculated to the cocoa bean and then fermented .
• a method for silage production As known in the art - Lactobacillus plantarum cells have been used for cocoa bean fermentation. In line of this - a herein relevant use of the Lactobacillus plantarum cells as described herein is use for cocoa bean fermentation - i.e. a method for cocoa bean fermentation, wherein a Lactobacillus plantarum composition as described herein is inoculated to the cocoa bean and then fermented .
• Lactobacillus plantarum cells have been used for silage production .
• the third aspect relates to a method for screening and isolating a novel Lactobacillus plantarum cell .
• a Lactobacillus plantarum cell capable of fulfilling the conditions of point (i) and (ii) of the first aspect is selected for.
• D- cycloserine resistance and ethanol resistance assays may be changed to make a alternative screening method that still ob- tains the main goals as described herein, i .e. a Lactobacillus plantarum cell that is capable of fulfilling the conditions of point (i) and (ii) of the first aspect.
• the term "functionally equivalent antibiotic” should be understood as an antibiotic with the same mode of action or the same target as D- cycloserine, such as e.g . other inhibitors of D-alanyl-D-alanine ligases, such as e.g . vancomycin and other inhibitors of D-alanine racemase, such as e.g . O-carbamoyl-D- serine, alaphosphin and the haloalanines.
• D- cycloserine such as e.g . other inhibitors of D-alanyl-D-alanine ligases, such as e.g . vancomycin and other inhibitors of D-alanine racemase, such as e.g . O-carbamoyl-D- serine, alaphosphin and the haloalanines.
• step (b) is the isolation of a novel Lactobacillus plantarum that is capable of fulfilling the conditions of point (i) and (ii) of the first aspect.
• a separate aspect of the invention relates to a Lactobacillus plantarum cell, which is capable of fulfilling the conditions of point (i) and (ii) of the first aspect and is obtainable by the screening method of the third aspect herein.
• Step (a) of the method for screening and isolating a novel Lactobacillus plantarum cell of the third aspect reads "selecting and isolating from a pool of individual Lactobacillus plantarum cells”.
• mutagenesis e.g . using a chemical mutagen or UV mutagenesis
• suitable mutagenesis e.g . using a chemical mutagen or UV mutagenesis
• the starting Lactobacillus plantarum cell CHCC14158 was subjected to mutagenesis (using D-cycloserine as selective agent) and from the created pool of individual Lactobacillus plantarum cells was subsequently selected the novel ethanol resistant cells CHCC14254 and CHCC14255 according to the selection method as described herein.
• D-cycloserine - such as e.g. Lactobacillus plantarum cells described in the article of Bron et al. (2002) as discussed above.
• Relevant Lactobacillus plantarum cells of the Bron et al . (2002) could then e.g. be subjected to a suitable mutagenesis and then selected for improved resistance towards ethanol as discussed herein.
• the medium is the known Grape Juice GJ-5 medium described in column 20, lines 10 to 20 of US7112346 (Chr. Hansen A/S) .
• the GJ-5 medium has the following composition :
• Grape juice concentrate 70.0 g
• this GJ-5 medium is a medium that is considered to be representative for a grape juice used for wine production.
• a grape juice concentrate is a standard well known ingredient of such a medium.
• the specific Grape juice concentrate may be supplied from different suppliers and independently of the specific supplier one will (within standard measurement uncertainty) get the same herein relevant result of cycloserine resistance for a herein relevant cell of interest.
• a Lactobacillus plantarum strain of interest is inoculated into 10 ml GJ-5 medium containing one of the following amounts of D-cycloserine : 0 Mg/ml, 10 g/ml, 20 Mg/ml, 30 ⁇ g/ml, 50 Mg/ml, 70 pg/ml, 100 Mg/ml, 150 ⁇ g/ml or 200 ⁇ g/ml of D- cycloserine.
• the strain is grown 24 hours at 18°C in the GJ-5 medium with the different concentrations of D-cycloserine.
• a Lactobacillus plantarum cell that has an increased resistance to D-cycloserine as discussed herein - is herein defined as a Lactobacillus plantarum cell, wherein the amount of D-cycloserine that reduces the OD 500 measured growth, after 24 hours growth at 18°C, with 50% in GJ-5 medium as compared to the growth in GJ-5 medium without D-cycloserine (i .e. with 0 g/ml D-cycloserine) is higher than 70 g/ml D-cycloserine.
• Cells that are capable of complying with this increased resistance to D-cycloserine criteria are herein defined as cells that are positively resistant to D-cycloserine in the D-cycloserine resistance assay of this example 1.
• the medium is the standard GJ-5 medium as used in Example 1 above.
• a Lactobacillus plantarum cell that has an improved resistance towards ethanol as discussed herein - is herein defined as a Lactobacillus plantarum cell that can grow to an OD 600 of at least 0.8 after 3 days incubation at 25°C in the GJ-5 medium with 11% ethanol .
• Cells that are capable of complying with this improved resistance towards ethanol criteria are herein defined as cells that are positively resistant to ethanol in the ethanol resistance assay of this example 2.
• EXAMPLE 3 Use of D-cycloserine to isolate mutants of Lactobacillus plantarum with improved resistance to high concentrations of ethanol Strains
• Lactobacillus plantarum CHCC14255 (D-cycloserine mutant of CHCC14158 isolated at 18°C in GJ-5)
• Lactobacillus plantarum CHCC14254 (D-cycloserine mutant of CHCC14158 isolated at 18°C in GJ-5)
• Lactobacillus plantarum CHCC14158 is a cell, wherein the amount of D-cycloserine that reduces the OD 600 measured growth with 50% in GJ-5 medium as compared to the growth in GJ-5 medium without D- cycloserine (i .e. with 0 g/ml D-cycloserine) is lower than 70 g/ml D-cycloserine, since the amount of D-cycloserine that reduced the growth with 50% was around 60 to 65 g/ml D-cycloserine - see e.g. Figure 1 herien.
• Lactobacillus plantarum CHCC14158 is not positively having increased resistance to D-cycloserine as defined in Example 1 above.
• Lactobacillus plantarum strain CHCC14158 was subjected to D-cycloserine pressure as described below.
• the D-cycloserine worked here as a selective agent to create a pool of mutant cells with increased resistance to D-cycloserine.
• CHCC14255 and CHCC14254 Two mutant derivatives of CHCC14158, designated CHCC14255 and CHCC14254, were significantly more resistant to high concentrations of ethanol than the mother strain when the growth was compared in GJ-5 at 25°C in the presence of 11, 12 and 13 % ethanol of parental strain CHCC14158 and two cycloserine resistant mutants CHCC14255 and CHCC14254.
• the two cycloserine resistant mutants CHCC14255 and CHCC14254 could both grow to an OD 600 of at least 0.8 after 3 days incubation at 25°C in the GJ-5 medium with 11% ethanol - for CHCC14255 the OD 600 was more than 1 - i .e. both strains were positively resistant to ethanol in the ethanol resistance assay of this example 2.
• the starting CHCC14158 strain could only grow to an OD 600 of around 0.65 - i .e. the starting CHCC14158 strain was not positively resistant to ethanol in the ethanol resistance assay of this example 2.
• the amount of D-cycloserine that reduces the OD 6D0 measured growth with 50% in GJ-5 medium as compared to the growth rate in GJ-5 medium without D-cycloserine was around 100 ⁇ g/ml D- cycloserine (see Figure 1 herein) .
• the amount of D-cycloserine that reduces the OD 600 measured growth with 50% in GJ-5 medium as compared to the growth in GJ-5 medium without D-cycloserine was around 100 pg/ml D- cycloserine (see Figure 1 herein) .
• UV mutagenesis was done on a Lactobacillus plantarum strain CHCC12396.
• Example 3 Screening for ethanol was done as in Example 3 above - however, after analysis of more than 100 different mutants/colonies it was not possible to select a mutant with improved resistance towards ethanol as defined in Example 2 above.

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